Regulation of gene expression is crucial for normal development and defects in this process can result in disease. A better understanding of the genetic elements that contribute to regulation of disease genes can reveal causes of disease and may spur development of novel treatments. Large-scale analyses of genomes, such as those initiated by the ENCODE project, have started to reveal the positions of many regulatory elements. However, in most cases it is not known which elements regulate any given gene. Understanding the relationships between regulatory elements and genes is complicated by the fact that the linear order of elements and genes along chromosomes often does not reflect functional relationships between them. For instance, regulatory elements may affect distal genes but not those located immediately next to them. Therefore, functional connections between genes and regulatory elements must be experimentally determined. This proposal is based on the hypothesis that regulatory elements physically associate with their target gene through formation of chromatin loops. We propose to test this hypothesis by mapping of looping interactions between genes and regulatory elements throughout the well-studied 1 % of the genome selected by the ENCODE consortium. We will detect chromatin loops using a unique approach, Chromosome Conformation Capture (3C) methodology. During the last 2 years we have developed a new high-throughput 3C application, called 5C (for 3C-carbon-copy) which employs microarrays or quantitative sequencing for detection of chromatin loops. We will further optimize 5C by analysis of the beta-globin locus (aim 1). We will employ 5C to identify chromatin loops between genes and regulatory elements throughout the well-studied ENCODE regions of the human genome (aim 2). For each gene we will identify distant elements such as enhancers that interact with its promoter. We will validate looping interactions by FISH (aim 3). We will test the function of looping elements using transient transfections and by integrating looping data with other data obtained by the ENCODE consortium such as histone modifications, DNasel hypersensitivity and target gene expression (aim 3). We will make looping data available through the UCSC genome browser. These studies will map the network of connections between genes and regulatory elements and will reveal new insights into the mechanisms that underlie long-range gene regulation.